Use of a biological material containing three-dimensional scaffolds of hyaluronic acid derivatives for the preparation of implants in arthroscopy and kit for instruments for implanting said biological material by arthroscopy

ABSTRACT

The invention relates to use of a biological material containing cells supported on three-dimensional scaffolds containing at least one hyaluronic acid derivative for the preparation of grafts suitable for application by arthroscopy, and a kit of surgical instruments for implanting said biological material by arthroscopy.

FIELD OF THE INVENTION

The present invention concerns the use of biological material containingcells supported on three-dimensional scaffolds which comprise at leastone hyaluronic acid derivative for the preparation of grafts suitablefor application by arthroscopy, and a kit for surgical instruments forimplanting said biological material by arthroscopy.

BACKGROUND OF THE INVENTION

The aim of joint cartilage repair is to restore the integrity of thejoint surface, reduce pain and prevent any further deterioration of thetissues.

Joint cartilage is a tissue which allows virtually frictionless movementof the joint. Its particular biological characteristics enable the jointto absorb forces at least five times greater than the body's weight. Thejoint cartilage, or hyaline, has a very limited capacity forself-repair, so the type of cartilage that is spontaneously regeneratedafter damage does not possess the same characteristics as the originaltissue. It is known as fibrocartilage and has no properties oflubrication or absorption of mechanical shock. The final phase ofhyaline cartilage degeneration is accompanied by pain and limitedmobility that may cause locking of the joint. In the long term, thedegenerative process may even cause the onset of complications such asosteoarthritis. In the most severe cases, the joint, usually the knee,has to be replaced with a metal prosthesis. This is a costly procedureand is not even permanent because many prostheses have to be changedafter about 10-15 years. For this reason, knee replacements are onlyperformed as a last resort in patients of under 50 years old. Jointcartilage lesions are currently treated by arthroscopic surgicaltechniques chiefly aimed at reducing pain, slowing down the degenerationprocess and, whenever possible, repairing the damage. Many methods havebeen applied to date to treat cartilage defects, and each of them hascertain disadvantages (T. Minas et al. “Current concepts in thetreatment of articular cartilage defects”, Orthopedics, June 1997, Vol.20 No. 6). One such technique involves trimming the margins of thecartilage defect, in other words, débridement of the edges of the lesionby removing any necrotic or diseased tissue. The technique ofstimulating the marrow consists in reaching areas of the subchondralbone tissue by abrasion or perforation, thus stimulating the formationof a fibrin clot containing pluripotent stem cells. The clot thendifferentiates and takes form, giving rise to fibrocartilage repairtissue. The resulting tissue does not, however, have the mechanicalproperties or physiological or structural characteristics of healthy,lasting joint cartilage.

Another technique consists in implanting a piece of periosteum orperichondrium tissue, taken for example from rib cartilage, into thedefect. Initially, this treatment triggers the development of hyalinecartilage, but the repair tissue does not take easily to the surroundinghealthy tissues, and subsequently becomes ossified. Recently, a team ofSwedish researchers devised an ex-vivo technique of grafting autologouschondrocytes, where chondrogenic cells are isolated from a smallcartilage biopsy, grown in vitro and then regrafted in the same subject(M. Brittberg, A. Lindahl, A. Nilsson: “Treatment of deep cartilagedefects in the knee with autologous chondrocyte transplantation”, N.Eng. J. Med: 1994, 331, 889-895). According to the authors, in theculture phase, the chondrocytes temporarily de-differentiate andmultiply under stimulation by suitable growth factors. Once transplantedto the damaged area, they recover their phenotype memory andconsequently re-differentiate into chondrocytes able to produce ahyaline-type cartilage matrix. The surgical procedure is actually rathercomplex. First of all, the operation requires open surgery. Moreover,the cartilage defect must be well located and covered by a lid ofperiosteum (taken in the course of the same operation). This must befixed to the cartilage tissue with a watertight seal of suture andfibrin (autologous or allogenic), so as to create a chamber into whichthe autologous cell suspension can be injected. Indeed, if the chamberis not perfectly sealed, the cells will leak out again and the operationwill have failed.

To summarise, the main disadvantages of this procedure are that theoperation is difficult to perform, the technique is invasive and theimplanted cells are not perfectly differentiated. Autologous andhomologous osteochondral grafts involve techniques that are surgicallyinvasive and complex and there is a risk of viral transmission with thelatter.

Other attempts at reconstructing the joint cartilage consist inimplanting synthetic scaffolds containing allogenic chondrocytes, andgrowth factors able to stimulate proliferation of the chondrocytes.

The most frequently used synthetic scaffolds are of collagen gel,polyanhydride, polyorthoester, polyglycolic acid and the copolymersthereof. The chief disadvantage of using said scaffolds is representedby an immune response directed towards the implanted material.

There are known chondrocyte cultures in gel-scaffolds constituted byagarose, hyaluronic acid, fibrin glue, collagen and alginate.

However, said cultures in gel do not provide suitable mechanicalstability to remain adhered to the site and allow the reconstruction ofthe cartilage structure.

Moreover, chondrocyte cultures in substances such as fibrindedifferentiate into cells that are apparently similar to fibroblasts.

Lastly, although the gels constituted by substances such as agaroseinduce chondrocyte redifferentiation, the use of this compound has notbeen approved for internal application in humans.

As previously described joint cartilage defects have also been treatedwith isolated chondrocyte suspensions in the absence of supportingscaffolds. It is thought, however, that chondrocytes lose theirviability and/or do not remain in the defect, and that they formfibrocartilage or islets of cartilage immersed in fibrous tissue (U.S.Pat. No. 5,723,331).

To overcome this problem, the Applicant has devised injectablecompositions containing chondrocytes or cells of bone marrow stromadispersed in a matrix containing at least one hyaluronic acid derivative(PCT patent application, publication No. WO00/37124).

As is known, hyaluronic acid plays a vital role in many biologicalprocesses, such as tissue hydration, proteoglycan organisation, celldifferentiation, proliferation and angiogenesis (J. Aigner et al. L.Biomed. Mater. Res. 1998, 42, 172-181).

Also known is the use of hyaluronic acid derivatives prepared asdescribed in EP patent No. 0216453 B1 for the preparation ofthree-dimensional scaffolds in the form of non-woven fabrics, membranes,sponges, granules, microspheres, tubes, gauzes, for the in vitro growthof stem and mesenchymal cells (PCT patent application publication No. WO97/18842), in the form of a nonwoven fabric associated with a perforatedmembrane for the growth in vitro of fibroblasts and keratinocytes (PCTpatent application No. WO 96/33750 and in the form of a nonwoven fabricfor the growth of chondrocytes (J. Aigner et al. L. Biomed. Mater Res.1998, 42, 172-181).

SUMMARY OF THE INVENTION

The Applicant has now found, surprisingly, that it is possible to use toeffect three-dimensional matrices based on hyaluronic acid derivativesas scaffolds for cellular material for implantation in patients atarthroscopy, and that the use of such matrices solves the above problemsinvolved in arthroscopic techniques.

The use of biocompatible and bioresorbable three-dimensional matricesbased on hyaluronic acid on which cells are grown represents a huge stepforward in arthroscopic techniques. Indeed, the cells begin todifferentiate into chondrocytes while they are still growing on thematrix, because of the three-dimensional stimulation and the presence ofsuitable growth factors. Cell differentiation with the production ofabundant hyaline matrix then continues in the lesion after grafting.

The fact that the cells are already mounted, before implantation, on athree-dimensional scaffold with hyaluronic acid's known properties ofbiocompatibility and bioresorption eliminates the need for a periostealflap to be sealed over the defect to form a watertight lid, because theonly covering the defect requires is one that will hold the graft inplace until it has taken to the surrounding cartilage tissue.

All that is required, therefore, is a fibrin sealant (autologous orallogenic), or another biological glue, for a limited length of time.The fact that there is no longer any need for a flap of periosteumrepresents another major advantage: the arthrotomy technique used in theSwedish model can be substituted with the less invasive and moreeconomical arthroscopy.

Subject of the present invention is therefore the use of a biologicalmaterial containing cells grown on three-dimensional matrices containingat least one hyaluronic acid derivative for the preparation ofimplantations suitable for application by the arthroscopic technique.

The present invention further relates to a kit of surgical instrumentsfor implanting the aforementioned biological material, said kitcomprising:

-   a) a sterilisation tray;-   b) a cannula with relative sterile valves to be used as a guide to    give access, during arthroscopy, to the set of instruments listed    hereafter;-   c) a mapper-sampler, constituted by a hollow, cylindrical tube, used    to circumscribe the cartilage lesion by creating a circular imprint,    and to take cartilage tissue in the same circular form and of the    same dimension as the imprint;-   d) a guide wire that is fixed with the aid of a drill to the centre    of the lesion, to guarantee stability to the cutter during use;-   e) a concave, hollow cutter-abrasor used to create, within the    margins of the imprint made by the mapper-sampler, the site in which    the bio-engineered cellular support will subsequently be implanted;-   f) a hollow plunger to be introduced into the mapper-sampler to push    the bio-engineered cellular support into the previously prepared    lesion site.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 show the results of Examples 3 and 4 insofar as itconcerns the recovery of cellular viability expressed as optical densityrespectively in the form of bar charts, where said optical density isreported on the co-ordinate at 470 nm expressed in absolute unitmeasured with a FLOW spectrophotometer.

FIG. 3(I) represents respectively a schematic lateral view FIG. 3(II) aschematic cross section view along A-A of the mappler sampler.

FIG. 4(I) represents respectively a schematic lateral view and FIG.4(II) a schematic cross section view along B-B of the hollow plunger.

FIG. 5(I) represents respectively a schematic lateral view, FIG. 5(II) aschematic cross section view along A-A, FIG. 5(III) a frontal view andFIG. 5(IV) a schematic and enlarged (3:1) frontal view of the concavehollow cutter-abrasor.

FIGS. 6(I) and 6(II) show a schematic representation of themapper-sampler, while creating an imprint around the lesion.

FIGS. 7(I) and 7(II) show a schematic representation of the cutterstabilised by the guide wire fixed to the centre of the lesion, whilefunctioning.

FIG. 8 shows a schematic representation of the mapper-sampler completewith the plunger as it places the biological material comprising cellsgrown on a three dimensional scaffold that has previously been cut outby the cutter,

DETAILED DESCRIPTION OF THE INVENTION

Of all the hyaluronic acid derivatives that can be used in thethree-dimensional scaffolds according to the present invention, thefollowing are the ones of choice:

hyaluronic acid esters wherein part or all of the carboxy functions areesterified with alcohols of the aliphatic, aromatic, arylaliphatic,cycloaliphatic or heterocyclic series (EP 0216453 B1);

crosslinked esters of hyaluronic acid wherein part or all of the carboxyfunctions are esterified with the alcoholic functions of the samepolysaccharide chain or other chains (EP 0341745 B1);

crosslinked esters of hyaluronic acid wherein part or all of the carboxyfunctions are esterified with polyalcohols of the aliphatic, aromatic,arylaliphatic, cycloaliphatic, heterocyclic series, generatingcrosslinking by means of spacer chains (EP 0265116 B1);

hemiesters of succinic acid or heavy metal salts of the hemiesters ofsuccinic acid with hyaluronic acid or with partial or total esters ofhyaluronic acid (WO 96/357207);

O-sulphated derivatives of hyaluronic acid (WO 95/25751) or N-sulphatedderivatives of hyaluronic acid (WO 98/01973);

Quaternary ammonium salts, for example salts with tetrabutylammonium andphenyltrimethylammonium, of hyaluronic acid or the derivatives thereofchosen from the group formed by N-sulphated hyaluronic acid, O-sulphatedhyaluronic acid, the hemiesters of succinic acid with hyaluronic acid,possibly partially salified with heavy metals;

O-sulphated or N-sulphated hyaluronic acid and the derivatives thereofcovalently bound to polyurethane (WO 99/43728).

The present three-dimensional scaffolds may also contain an associationof several kinds of hyaluronic acid derivatives, and may be in variousforms, such as nonwoven fabric as described in U.S. Pat. No. 5,520,916,meshes according to patent No. EP216453B1, perforated membranes asdescribed in EP462426B1 unperforated membranes as in EP 216453B1, andsponges as described in EP 216453B1.

Such matrices may also include associations of natural, semisynthetic orsynthetic polymers.

Natural polymers that can be used according to the present inventionare, for example, collagen, co-precipitates of collagen andglycosaminoglycans, cellulose, polysaccharides in the form of gels suchas chitin, chitosan, pectin or pectic acid, agar, agarose, xanthane,gellan, alginic acid or alginates, polymannan or polyglycans, starch,natural gums.

The semisynthetic polymers may be, for example, chosen from the groupconsisting of collagen crosslinked with agents such as aldehydes orprecursors of thereof, dicarboxylic acids or their halogenides,diamines, derivatives of cellulose, hyaluronic acid, chitin or chitosan,gellan, xanthane, pectin or pectic acid, polyglycans, polymannan, agar,agarose, natural gum and glycosaminoglycans.

Lastly, examples of synthetic polymers that can be used are polylacticacid, polyglycolic acid copolymers or derivatives thereof, polydioxanes,polyphosphazenes, polysulphonic resins, polyurethanes and PTFE.

The present three-dimensional scaffolds may also includepharmaceutically or biologically active ingredients, such asanti-inflammatory agents, antibiotics, growth factors, antimicotics,antimicrobial and antiviral agents.

The cells used to prepare the present biological material were chosenfrom chondrocytes, osteocytes, mesenchymal cells and stem cells.

The cell culture process used to make the biological material accordingto the invention is that described by Aigner et al, “Cartilage tissueengineering with novel unwoven structured biomaterial based onhyaluronic acid benzyl ester”, J. Biomed. Mat. Res. 1998, 42(2),172-181.

From seven days after seeding on the three-dimensional scaffold based onhyaluronic acid derivatives, and preferably after the fourteenth day,the biological material is ready to be grafted.

The present biological material may be reduced to the size of thecartilage defect with a packer as used in mosaicplasty during the actualgrafting process, that is, during surgery immediately beforeintroduction of the surgical instrument. Alternatively, the material canbe passed through a cannula, an instrument that is commonly used inarthroscopy or by using the surgical instruments of the kit ofarthroscopy further subject of the present invention.

The biological material according to the invention may also be used toprepare both autologous grafts and allogenic grafts suitable forapplication by arthroscopic techniques.

Another advantage of the present invention lies in the fact that saidbiological material can be cryopreserved in order to preserve itscharacteristics of cell viability ready for implantation to be performedat a future time.

The kit of instruments for implanting biological material to be used forarthroscopy according to the present invention was constructed usingmaterials with the preferred characteristic listed below:

The cannula previously listed at item (b) used as a guide to give accessin arthroscopy to the set of instruments listed hereafter andrepresented in FIGS. 6(I), 6(II), 8 (indicated with the number 5), is inAise 316 steel and presents an inside diameter of 11.5 mm and is 111 mmlong. In the aforementioned figures the sterile valves are notindicated.

The mapper-sampler previously listed at item (c), being used tocircumscribe the cartilage lesion by creating a circular imprint and tocut out and remove a piece of cartilage tissue of the same shape andsize, and represented in the FIGS. 3, 6(I), 6(II) and 8 (indicated withthe number 1) is a cannula in Aise 316 medical steel, 155 mm long, withan outer diameter of 10.5 mm and an inside diameter of 9 mm. The mapplersampler 1 in the kit according to the present invention is a hollowcylinder large enough to hold the plunger and is further characterisedby having a concave tip like the mapper sampler and a control system bywhich the pressure exercised by the advancing plunger can beinterrupted.

The guide wire previously listed at item (d), fixed with the aid of adrill to the centre of the lesion to stabilise the cutter during use,and represented in FIG. 7(I) (indicated with the number 3) has thediameter of 1 mm.

The concave hollow cutter-abrasor previously listed at item (e), used tocreate within the margins of the imprint left by the mapper 1 the sitein which the biological material to be used in arthroscopy according tothe present invention for subsequent grafting and represented in FIGS. 5and 7(I) and 7(II) (indicated with 4) is in Aise 316 medical steel, is162.5 mm long with an inside diameter of 1.2 mm and an outer diameter of9.5 mm.

The blades of the cutter 4 are concave so that they produce convexsurfaces. The hollow plunger previously listed at item (e) that isintroduced into the mapper-sampler 1 to push the biological material tobe used in arthroscopy according to the present invention into thepreviously prepared lesion site and represented in FIGS. 4, 6(I), 6(II)and 8, (indicated with the number 2), has a diameter of 5 mm is a hollowcylinder large enough to hold a guide wire and that it has a concave tiplike the mapper.

The use of said instruments enables the following operative technique tobe performed:

-   A) a pneumatic tourniquet is placed around the proximal area of the    limb the lesion area is identified by arthroscopy,-   B) a needle is used to identify a point of entry directly above the    lesion,-   C) the skin is cut with a scalpel and a cannula 5 is introduced    through the point of entry, through which the mapper-cutter 1 will    be introduced to make a circular imprint within the lesion, 9 mm in    diameter (mapping operation);-   D) through the mapper 1 passes the concave, hollow plunger 2, and    into this is introduced, in turn, the guide wire of 1 mm diameter,    fixed with the help of a drill to the centre of the imprint. This    guide wire will serve to stabilise the subsequent cutting operation;-   E) both the mapper 1 and the plunger 2 are removed and the concave    cutter 4, of the same size as the mapper (1) used earlier, is    introduced. Holding the cutter perpendicular to the lesion, the    latter is shaped, stopping at the distal point marked on the cutter;-   F) using the mapper-sampler 1, the biological material of the    dimensions ‘mapped’ earlier containing cells grown on the    bio-engineered, three-dimensional scaffold is prepared and    introduced through the cannula (1) complete with its concave plunger    2, thus enabling the biological material to be applied to the    lesion,-   G) the hollow, concave plunger 2 pushes the scaffold out of the    mapper 1 into the convex hollow of the lesion;-   I) the joint is repeatedly flexed and straightened to check the    stability of the graft,-   J) The pneumatic tourniquet is released and the arthroscopic    apparatus (optical and the cannula 5) is removed.

The kit according to the present invention may be used also forImplanting the three-dimensional scaffolds containing autologous and/orallogenic cells, which can be constituted by natural, semisynthetic orsynthetic polymers, free from hyaluronic acid derivatives.

The natural polymers are chosen from the group formed by collagen,coprecipitates of collagen and glycosaminoglycans, cellulose,polysaccharides in the form of gels such as chitin, chitosan, pectin orpectic acid, agar, agarose, xanthane, gellan, alginic acid or alginates,polymannans or polyglycans, starch and natural gums.

The following are the semisynthetic polymers of choice: collagencrosslinked with agents such as aldehydes or precursors thereof,dicarboxylic acids or their halides, diamines, derivatives of cellulose,hyaluronic acid, chitin, chitosan, gellan, xanthane, pectin or pecticacid, polyglycans, polymannan, agar, agarose, natural gum andglycosaminoglycans.

The synthetic polymers are chosen from the group formed by polylacticacid, polyglycolic acid, the copolymers or derivatives thereof,polydioxanes, polyphosphazenes, polysulphonic resins, polyurethanes andPTFE.

Moreover, the three-dimensional scaffolds according to the presentinvention may contain, besides cells, pharmaceutically or biologicallyactive substances such as anti-inflammatory agents, antibiotics, growthfactors, antimicotic or antiviral agents, and they may be cryopreservedto preserve their characteristics of cell viability ready for subsequentgrafting by arthroscopy using the set of instruments claimed hereafter.

The following examples are for illustrative purposes and do not limitthe scope of the present invention.

EXAMPLE 1 Preparation of the Biological Material

The cell culture process is described in Brun et al., “Chondrocyteaggregation and reorganisation into three dimensional scaffolds”, J.Biomed. Mater. Res. 1998, 46, 337-346. Cartilage tissue taken from anon-weight-bearing area marginal to the lesion is detached by treatmentwith type-II collagenase, and the cells thus obtained are seeded indishes containing HAM's F12 medium supplemented with foetal calf serum,1% streptomycin-penicillin, 1% glutamine and with the following trophicfactors, each of which in a quantity ranging between 1 and 10 ng/ml: TGFβ1, recombinant human EGF, recombinant human insulin and recombinanthuman bFGF.

The cells are grown in vitro from one to four serial passages, thenseeded on HYALOGRAFT®C (a non-woven fabric based on HYAFF®11t—totalbenzyl ester of hyaluronic acid), at a cell density of between 0.5×10⁶and 4×10⁶ cells/cm², and the culture medium described above. At eachchange of medium (2-10 ml every 48-72 hours) 50 μg/ml of ascorbic acidis added.

EXAMPLE 2 Valuation of Cell Viability by the MTT Test

Cell viability of the biological material is determined by incorporationof the vital MTT dye (F. Dezinot, R. Lang “Rapid calorimetric assay forcell growth and is survival. Modification to the tetrazolium dyeprocedure giving improved sensitivity and reliability” J. Immunol.Methods, 1986, 22 (89), 271-277). A solution prepared by dissolving 0.5mg/ml of MTT in phosphate buffer, pH 7.2 (PBS) is added to the testmaterial and placed in an incubator set at 37° C. for 4 hours. Onceincubation is complete, the MTT solution is aspirated, the materialwashed several times with PBS, after which 5 ml of extracting solutionconstituted by 10% dimethylsulphoxide in isopropyl alcohol is added.After centrifugation, the absorbance of the supernatant is determined bya spectrophotometric reading at 470 nm.

EXAMPLE 3 Verification of Cell Viability Recovery After Passage Througha Cannula

The MTT test as described in Example 2 is used to verify cell viabilityrecovery after passage through a cannula, an instrument commonly used inarthroscopy and used here to place the biological material.

The results are reported in FIG. 1, which shows cell viability recoveryrelative to a bio-engineered cartilage construction (Hyalograft®C,dimensions 2×2 cm), prepared as described in Example 1. After 72 hoursin its packaging, that is, in conditions of maximum metabolic stress,the biomaterial was gently extruded through a cannula with a diameter of9 mm. It was found that the passage through the hollow of the cannuladoes not modify the cell viability of the present material.

EXAMPLE 4

Verification of cell viability recovery after packing with amosaicplasty packer Once again using the MTT test as described above inExample 2, the viability of a bio-engineered construction reduced to thedesired dimensions with a packing instrument as used in mosaicplasty,thus mimicking the conditions in which bio-engineered material isreduced in size to fit a cartilage defect for grafting, that is, duringsurgery, immediately before its insertion with the surgical instrument.

In this experiment, a cellular construction was kept in its packaginguntil the expiry date of the product (72 hours), then divided intosections 9 mm In diameter with a packer used in mosaicplasty. Theviability of the single pieces was normalised by surface unit, andcompared with the residue biological material.

FIG. 2 shows the results of this experiment.

The cell viability tests described above demonstrated that theapplication by arthroscopy of the present material, that is, biologicalconstructs that have reached their expiry deadline (72 hours) inconditions of maximum metabolic stress, does not influence theirbiological qualities. This is regardless of the type of surgicalinstrument used to reduce the material and the packaging conditions ofthe product.

The invention being thus described, it is clear that the materials andmethods used can be modified in various ways. Such modifications are notto be considered as divergences from the spirit and purpose of theinvention and any such modification which would appear evident to anexpert in the field comes within the scope of the following claims:

1. A method for the treatment of a cartilage lesion by implantation of agraft at the cartilage lesion site by arthroscopic techniques, saidmethod comprising the steps of: preparing an autologous and/or allogenicgraft comprising a biological material suitable for implantation byarthroscopic techniques, by growing autologous and/or allogenic cellschosen from the group consisting of chondrocytes, osteocytes,mesenchymal cells, and stem cells onto three-dimensional scaffolds, fora period of time of seven to fourteen days, wherein said scaffoldscomprise at least one hyaluronic acid derivative that is an ester ofhyaluronic acid, wherein part or all of the carboxy functions of saidhyaluronic acid are esterified with alcohols of the arylaliphaticseries; circumscribing the cartilage lesion by creating a circularimprint by means of a mapper-sampler; cutting out and removing a pieceof damaged cartilage tissue having the same size and shape as theimprint; preparing the so obtained biological material for arthroscopicimplantation by reducing said biological material by means of themapper-sampler to the size and shape of said imprint; introducing the soreduced biological material into a cannula and applying the same to thecartilage lesion site.
 2. The method according to claim 1, furthercomprising the step of adding one or more pharmaceutically orbiologically active ingredients onto said three-dimensional scaffoldsbefore the step of growing said autologous and/or allogenic cells. 3.The method according to claim 1, further comprising the cryopreservationof the said biological material before the step of preparing it forarthroscopic implantation in order to preserve the characteristics ofcell viability ready for grafts to be performed at a future time.
 4. Themethod according to claim 1, wherein said three-dimensional scaffoldsare in the form of a non-woven fabric.
 5. The method according to claim1, wherein said three-dimensional scaffolds comprise a total benzylester of hyaluronic acid.
 6. The method according to claim 1, whereinsaid three-dimensional scaffolds further comprise one or morepharmaceutically or biologically active ingredients.
 7. The methodaccording to claim 1, wherein said three-dimensional scaffolds consistof an ester of hyaluronic acid, wherein part or all of the carboxyfunctions are esterified with alcohols of the arylaliphatic series. 8.The method according to claim 7, wherein said three-dimensionalscaffolds consist of a total benzyl ester of hyaluronic acid.
 9. Themethod according to claim 7, wherein said three-dimensional scaffoldsare in the form of a non-woven fabric.
 10. The method according to claim7, wherein said three-dimensional scaffolds consist of an ester ofhyaluronic acid, wherein part or all of the carboxy functions areesterified with alcohols of the arylaliphatic series.
 11. The methodaccording to claim 10, wherein said three-dimensional scaffolds consistof a total benzyl ester of hyaluronic acid.